Project Summary The present project has the goal of developing flow cytometry methods to analyze glia and neurons within enzymatically dissociated cells from cryopreserved human cortex, and to simultaneously isolate cell-specific extracelleular vesicles(EVs)/exosomes from the same tissue samples. As a proof of concept initial hypothesis, we expect to observe stage-mediated tau increases in AD neurons and glia, and in EVs from the same samples. An extensive literature documents the importance of synaptic tau propagation in Alzheimer's disease and other tauopathies tau immunotherapy has shown efficacy in more than 13 studies. More recent studies demonstrate a major role for microglia and possibly astrocytes in the spread of neuronal tau pathology, but to our knowledge, no approach exists to analyze multiple brain cell types from the same human brain. Samples will come from our bank, developed over decades, of more than 350 postmortem AD and aged control cortex samples that are cryopreserved at the time of autopsy. A tauopathy mouse model that expresses human tau will also be studied. Aim 1 will optimize flow cytometry methods to identify and isolate neurons, astrocytes and microglia in dissociated cells from cryopreserved AD cortex. Aim 2 will isolate exosome/EVs from each AD cortical sample at the time of dissociation, and isolate tau-bearing EVs of neuronal, astrocyte and microglial origin. This proposal will directly address the contribution of the major brain cell types to tau progression in AD, at the same time considering the role of exosomes from each cell type. The proposed protocols are highly novel and will maximize the use of postmortem samples and our flow cytometry expertise.